The present invention describes sugar derivatives of indolocarbazoles which exhibit topoisomerase-I activity and are useful in inhibiting the proliferation of tumor cells.
Topoisomerases are vital nuclear enzymes which function to resolve topological dilemmas in DNA, such as overwinding, underwinding and catenation, which normally arise during replication, transcription and perhaps other DNA processes. These enzymes allow DNA to relax by forming enzyme-bridged strand breaks that act as transient gates or pivotal points for the passage of other DNA strands. Topoisomerase-targeting drugs appear to interfere with this breakage-reunion reaction of DNA topoisomerases. In the presence of topoisomerase active agents, an aborted reaction intermediate termed a xe2x80x98cleavable complexxe2x80x99 accumulates and results in replication/transcription arrest, which ultimately leads to cell death. The development of topoisomerase I active agents therefore offers a new approach to the multi-regimental arsenal of therapies currently used in the clinic for the treatment of cancer.
An article in Cancer Chemother. Pharmacol [1994, 34 (suppl): S 41-S 45] discusses topoisomerase I active compounds that have been found to be effective clinical anti-tumor agents. Structurally these clinical candidates are related to the alkaloid camptothecin.
Indolo[2,3-a]carbazole alkaloids such as rebeccamycin (U.S. Pat. Nos. 4,487,925 and 4,552,842) and its water-soluble, clinically-active analog, 6-(2-diethylaminoethyl)rebeccamycin (U.S. Pat. No. 4,785,085), are useful antitumor agents which target DNA. Furthermore, fluoroindolocarbazoles have been disclosed in WO 98/07433 to act as antineoplastic agents with topoisomerase I inhibitory activity.
Indolo[2,3-a]carbazole derivatives related to the Rebeccarnycin class are disclosed (EP Appl. 0 545 195 B1 and 0,602,597 A2; Cancer Research 1993, 53, 490-494; ibid 1995, 55, 1310-1315) and claimed to exhibit anti-tumor activity. However, the major mechanism of action of these derivatives may not be like camptothecin, which acts as a topoisomerase I poison. Other indolocarbazoles related to those mentioned above are disclosed in WO 95/30682 and are claimed to exhibit anti-tumor activity.
Hudkins, et al. disclosed a series of fused pyrrolocarbazoles (WO 96/11933 and U.S. Pat. No. 5,475,110) and showed in vitro biological data such as inhibition of neuronal choline acetyltransferase (CHAT) and protein kinase C (PKC) inhibition for some compounds. U.S. Pat. No. 5,468,849 discloses certain fluororebeccamycin analogs as useful antitumor agents, along with a process for their production by fluorotryptophan analog feeding of a rebeccamycin-producing strain of Saccharothrix aerocolonigenes, particularly Saccharothrix aerocolonigenes C38,383-RK2 (ATCC39243). Glicksman, et al. disclose indolocarbazole alkaloids (U.S. Pat. No. 5,468,872) which are different in structure from those of the present invention. Kojiri, et al. disclose indolopyrrolocarbazoles having a dissacharide substituent (WO 96/04293) which are not related to the anhydrosugar indolocarbazoles. Weinreb, et al (Heterocycles 1984, 21, 309) and Kleinschroth, et al (U.S. Pat. No. 5, 043,335) have disclosed indolopyrrolocarbazole derivatives with a bridging furan moiety and McCombie, et al. (Bioorg. Med. Chem. Lett. 1993, 3, 1537) have reported a more functionalized bridged furan. Similarly, Wood, et al. have reported the total synthesis of (+)-K252a (J. Am. Chem. Soc. 1995, 117, 10413), a related, naturally-occuring indolocarbazole alkaloid which has demonstrated PKC inhibitory activity.
Danishefsky, et al., during the course of their first total synthesis of staurosporine (J. Am. Chem. Soc. 1996, 118, 2825), describe the synthesis of an intermediate N12, N13-bridged indolopyrrolocarbazole. Indolocarbazole derivatives with the nitrogens linked by a three-atom bridge have been reported to be potent PKC inhibitors (S.F. Vice, et al. Bioorg. Med. Chem.Lett. 1994, 4, 1333). The synthesis of simple indolocarbazole derivatives with C1xe2x80x2, C-5xe2x80x2-bridging or C1xe2x80x2, C3xe2x80x2-bridging glycosides have also been reported in the literature (B. M. Stolz, J. L. Wood Tetrahedron Lett. 1995, 36, 8543, B. B. Shankar, S. W. McCombie Tetrahedron Lett. 1994, 35, 3005, respectively). Prudhomme, et al. disclose a series of antitumor indolocarbazoles derived from rebeccamycin which exhibit a carbohydrate attached to the two indole nitrogens, and reported their cytotoxicity and their topoisomerase I and PKC inhibitory activities to be in the millimolar to micromolar range (Bioorg. Med. Chem. 1998, 6, 1597). There is yet a need for novel and potent cytotoxic compounds useful for inhibiting topoisomerase I activity. 
Thus according to a first embodiment of the first aspect of the present invention are provided compounds of Formula (I) and pharmaceutically acceptable salts and solvates thereof, useful for inhibiting topoisomerase I and the proliferation of tumor cells
wherein:
R is hydrogen, OH, OC1-7alkyl, NH2, N(C1-3alkyl)2 or C1-7alkyl, wherein said C1-7alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, OR9 and NR9R10;
Q is O, S, CH2 or NR5a;
R5 and R5aare each independently selected from the group consisting of 
xe2x80x83provided that
if Q is NR5a, then either R5 or R5a must be hydrogen;
R1, R2, R3 and R4 are each independently selected from the group consisting of hydrogen, C1-7alkyl, C3-7cycloalkyl, halogen, azido, NR9R10, NHC(O)NR9R10, NHC(O)OR9, C(O)OR9, SR9 and OR9, wherein said C1-7 alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, OR9, SR9 and NR9R10;
or R1 and R2 together form xe2x95x90Nxe2x80x94OH, xe2x95x90O or xe2x80x94NR9R10;
or R3 and R4 together form xe2x95x90Nxe2x80x94OH, xe2x95x90O or xe2x80x94NR9R10;
W is selected from the group consisting of hydrogen, C1-7alkyl, C3-7cycloalkyl, halogen, azido, NR9R10, NHC(O)NR9R10, NHC(O)OR9, Nxe2x80x94OH, O and OR9, wherein said C1-7alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, OR9 and NR9R10;
R7 and R8 are independently OH or H or together form xe2x95x90O;
R9 and R10 are independently selected from the group consisting of hydrogen, C1-7alkyl and C3-7cycloalkyl, wherein said C1-7alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, OH, Oxe2x80x94C1-7alkyl, NH2 and N(C1-3alkyl)2; or
R9 and R10 together with the nitrogen atom to which they are attached form a non-aromatic 5-8 membered heterocycle containing one or two of the same or different heteroatoms selected from the group consisting of O, N and S; and
X1, X1xe2x80x2, X2 and X2xe2x80x2 are independently selected from the group consisting of hydrogen, halogen, cyano, OR9, xe2x80x94CF3, alkylcarbonyl, C-1-7alkyl, nitro, NR9R10, SR9 and C(O)OR9; wherein said C1-7alkyl is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, OR9, SR9 and NR9R10.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5a is not H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5a is formula (C) or (A).
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5 is formula (A).
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5 is formula (B).
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5 is formula (C).
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R5 is formula (D).
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein Q is NR5a and R5a is H or wherein Q is S.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R is H, OH or NH2.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R is H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R7 and R8 together are xe2x95x90O.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein X2xe2x80x2 and X2 are each F and X1 and X1xe2x80x2 are each H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein X2 is F and X2xe2x80x2, X1 and X1xe2x80x2 are each H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein X2xe2x80x2 is F and X2, X1 and X1xe2x80x2 are each H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein X2xe2x80x2, X2, X1 and X1xe2x80x2 are each F.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein X2xe2x80x2 and X2 are each H and X1 and X1xe2x80x2 are each F.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein R1, R2, R3 and R4 are independently selected from the group consisting of H, F and OR9 wherein R9 is H.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein W is fluorine.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein the bond attaching R5 to N is in the xcex2 designation when R5 is not hydrogen.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein the bond attaching R5a to N is in the xcex2 designation when R5a is not hydrogen.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein the bond attaching R5 to N is in the xcex1 designation when R5 is not hydrogen.
According to another embodiment of the first aspect of the present invention are provided compounds of Formula (I) wherein the bond attaching R5a to N is in the xcex1 designation when R5a is not hydrogen.
Other embodiments of the first aspect of the present invention provide compounds of Formula (I) comprising two or more of the above embodiments of the first aspect suitably combined.
Embodiments of a second aspect of the present invention provide a method for inhibiting tumor growth in a mammalian host which comprises the administration to said host of a tumor-growth inhibiting amount of a compound of the present invention as defined herein.
Embodiments of a third aspect of the present invention provide a method for inhibiting tumor growth in a mammalian host, particularly a human host, comprising the administration to said host of a tumor-growth inhibiting amount of a pharmaceutical formulation of a compound of the present invention as defined herein.
Other embodiments and aspects of the invention will be apparent according to the description provided below.
The description of the invention herein should be construed in congruity with the laws and principals of chemical bonding. An embodiment or aspect which depends from another embodiment or aspect, will describe only the variables having values and provisos that differ from the embodiment or aspect from which it depends. Thus, for example, an embodiment which reads xe2x80x9cthe compound of formula (I) according to the nth aspect of the invention, wherein W is Cxe2x80x9d should be read to include all remaining variables with values defined in the nth aspect and should be read to further include all the provisos, unless otherwise indicated, pertaining to each and every variable in the nth aspect. Where a variable is defined as having a value of zero, it is understood that the bond attached to said variable should be removed. For example, if n=0 and Rxe2x80x94Xxe2x80x94Vn wherein n can be 0 or 1, then it is understood that the structure described is Rxe2x80x94X not Rxe2x80x94Xxe2x80x94.
The numbers in the subscript after the symbol xe2x80x9cCxe2x80x9d define the number of carbon atoms a particular group can contain. For example xe2x80x9cC1-7alkylxe2x80x9d means a straight or branched saturated carbon chain having from one to seven carbon atoms including without limitation groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-pentyl, isopentyl, n-hexyl and n-heptyl. The term xe2x80x9chalogenxe2x80x9d includes fluoro, chloro, bromo and iodo.
It is to be understood that the present invention includes any and all possible stereoisomers, geometric isomers, diastereoisomers, enantiomers, anomers and optical isomers, unless a particular description specifies otherwise.
The compounds of this invention can exist in the form of pharmaceutically acceptable salts. Such salts include addition salts with inorganic acids such as, for example, hydrochloric acid and sulfuric acid, and with organic acids such as, for example, acetic acid, citric acid, methanesulfonic acid, toluenesulfonic acid, tartaric acid and maleic acid. Further, in case the compounds of this invention contain an acidic group, the acidic group can exist in the form of alkali metal salts such as, for example, a potassium salt and a sodium salt; alkaline earth metal salts such as, for example, a magnesium salt and a calcium salt; and salts with organic bases such as a triethylammonium salt and an arginine salt. The compounds of the present invention may be hydrated or non-hydrated.
The compounds of this invention can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. The compounds of this invention may also be administered intravenously, intraperitoneally, subcutaneously, or intramuscularly, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. The compounds can be administered alone, but generally will be administered with a pharmaceutical carrier selected upon the basis of the chosen route of administration and standard pharmaceutical practice. Compounds of this invention can also be administered in intranasal form by topical use of suitable intranasal vehicles, or by transdermal routes, using transdermal skin patches. When compounds of this invention are administered transdermally the dosage will be continuous throughout the dosage regimen.
One aspect of the present invention involves administration of the compounds of the present invention, or pharmaceutically acceptable salts or solvates thereof, to a mammal implanted with a tumor or susceptible to cancer formation. In general the compound would be given in a dose range of from about 0.01 mg/kg to about the MTD (maximum tolerated dose). The dosage and dosage regimen and scheduling of a compounds of the present invention must in each case be carefully adjusted, utilizing sound professional judgment and considering the age, weight and condition of the recipient, the route of administration and the nature and extent of the cancer disease condition. The term xe2x80x9csystemic administrationxe2x80x9d as used herein refers to oral sublingual, buccal, transnasal, transderrnal, rectal, intramascular, intravenous, intraventricular, intrathecal, and subcutaneous routes. In accordance with good clinical practice, it is preferred to administer the instant compounds at a concentration level which will produce effective beneficial effects without causing any harmful or untoward side effects.
Procedures for the preparation of compounds of the present invention compounds are illustrated in Schemes 1-4: 
Compounds of the present invention and their methods of preparation are further described by the following non-limiting examples.
Several intermediate compounds as well as other conventional starting materials, used in the preparation of final products of compounds of the present invention, were generally known in the literature (WO 9807433) or were commercially available. Representative syntheses of some of these compounds are nevertheless provided hereinbelow.
The variables described in the above schemes have the same values as described in Formula (I), except for V in the above schemes which equals O, and W in the above schemes which equals R7 and R8 according to Formula (I). The benzyl (Bn) protecting group is illustrated as a particular moiety to xe2x80x9cprotectxe2x80x9d a hydroxyl functionality, but other suitable protecting groups well known to one skilled in the art may be used in lieu of benzyl or the like. Such suitable protecting groups are adequately described in Green""s Protecting Groups in Organic Synthesis (John Wiley and Sons, New York).
The starting materials in Scheme I, III and IV are glycosylated indolopyrrolocarbazoles and their preparation is described in WO9807433. Selective derivatization at the 6xe2x80x2-position may be achieved directly from compound of Formula 15 (R1xe2x95x90H, R2xe2x95x90OH), wherein all sugar hydroxyl groups are unprotected. Such chemoselective activation of the 6xe2x80x2-hydroxyl group to a good leaving group, such as mesylate or halide, is done in the presence of a base like triethylamine or pyridine using reagents for activation of a hydroxyl group to a good leaving group, such as methanesulfonyl chloride and others typically so used by one skillled in the art. More particular conditions are pyridine and methanesulfonyl chloride at 0xc2x0 C.
Intramolecular nucleophilic displacement of the 6xe2x80x2-mesylate, or other such leaving groups, by the 3 xe2x80x2-hydroxyl moiety is catalyzed by a base like triethylamine or Hunig""s base, but more particularly fluoride ion (such as tetrabutylammonium fluoride). Typical solvents for this reaction are, but are not limited to, DMSO, NMP, THF, N,N-dimethylimidazolidinone or DMF at temperatures from 0xc2x0 C. to 150xc2x0 C., but more particularly at 85xc2x0 C. The product of such an intramolecular displacement of a 6xe2x80x2-leaving group by the nucleophilic 3xe2x80x2-hydroxyl group is the 3xe2x80x2,6xe2x80x2-anhydrosugar derivative of the indolopyrrolocarbazole of Formula (I).
Other methods for the synthesis of 3xe2x80x2,6xe2x80x2-anhydrosugar analogs of Formula (I) from compounds of Formula 15 employ typical conditions of the Mitsunobu reaction, wherein the combination of triphenylphosphine (TPP) and diisopropylazodicarboxylate (DIAD) is used to activate the 6xe2x80x2-hydoxyl group towards intramolecular displacement by the 3xe2x80x2-hydroxyl group. Typical solvents for this reaction are, but not limited to, benzene, toluene, dioxane, more particularly THF or pyridine at temperatures from xe2x88x9215xc2x0 C. to 80xc2x0 C, and more particularly room temperature. Other reagents and/or combinations similar to TPP and DIAD may also be employed, such as diethylazodicarboxylate (DEAD) and TPP, or tri(O)-tolylphosphine, as well as TMAD and tributylphosphine and ADDP and trimethylphosphine, as well as combinations thereof. Additives such as 4-dimethylaminopyridine (4-DMAP) and imidazole may also be used to improve the yield and accelerate the rate of this reaction. For example, more particular is the use of 4-dimethylaminopyridine (4-DMAP) in combination with TPP and DIAD in such solvents as THF or the like. The use of the additive 4-DMAP is preferred and a preferred substrate for this modification is the glycosylated indolopyrollocarbozole of Formula 20 wherin R1xe2x95x90R2xe2x95x90H and the hydroxyl moiety at the 2xe2x80x2 position is preferably protected as the benzyl ether. In such a substrate, the 6xe2x80x2-hydroxyl moiety is activated towards intramolecular nucleophilic displacement by the free 3xe2x80x2-hydroxyl moiety, which is the only other hydroxyl moiety present.
Another method for the synthesis of 3xe2x80x2,6xe2x80x2-anhydrosugar analogs of Formula (I) employs as a substrate the 6xe2x80x2-fluoro sugar analog of Formulas 16A or 16B. Under appropriate conditions, hydrazine or ammonium acetate in ethanol at reflux, the 6xe2x80x2-fluorine can serve as a leaving group towards the intramolecular nucleophilic displacement by the 3xe2x80x2-hydroxyl moiety giving products of Formula (I). These examples, wherein fluorine serves a leaving group, would not generally be expected by one skilled in the art.
Rearrangement of the 3xe2x80x2,6xe2x80x2-anhydrosugar moiety under condiditons such as those typically used to convert the imide nitrogen to its free NH form (from its protected precursor) or to its N-O-benzyl form (from the same) can also serve to induce a novel rearrangement to yield glycosylated analogs such as the bicyclo [3.3.0] analog shown in Formula 17. Ethanol can be used as the solvent and at a reaction temperature including, but not limited to, that achieved by reflux of the solvent.
The synthesis of 3xe2x80x2,4xe2x80x2 and 2xe2x80x2,3xe2x80x2 anhydrosugar analogs of Formulas 18-19 proceed from their corresponding 3xe2x80x2,4xe2x80x2 and 2xe2x80x2,3xe2x80x2 trans-diol sugar analogs. Methods for the synthesis of compounds of Formulas 18-19 via dehydration utilize similar conditions employed for the synthesis of the 3xe2x80x2,6xe2x80x2-anhydrosugar analogs of Formula (I) (vide supra). A particular method for the synthesis of compounds of Formulas 18 and 19 is the Mitsunobu reaction using TPP and DIAD in THF, although other reagent combinations and solvents delineated above for the synthesis of compounds of Formula (I) may also be employed. A particular temperature for the synthesis of 3 xe2x80x2,4xe2x80x2 and 2xe2x80x2,3 xe2x80x2-anhydrosugar analogs of 18-19 is room temperature to 50xc2x0 C.
All anhydrous reactions were performed under an atmosphere of nitrogen or argon using either commercially available dry solvents or freshly distilled solvents. Melting points were determined in an open capillary tube with a Thomas-Hoover melting point apparatus and are uncorrected. Column chromatography was performed using EM Science silica gel 60 (230-400 mesh) with the designated solvent system as eluant. Thin-layer chromatography was done on E. Merck silica gel 60 F254 plates (0.5 mm). HPLC purity determinations were done using either a Shimadzu LC-10AS with a SPD-10AV UV-Vis detector and one of the folowing columns; YMC Combiscreen ODS-A (4.6xc3x9750 mm), or HP Zorbax SB-C18 (4.6xc3x97750 mm); or, an HP 1090 DR5 with a diode array detector and a Waters Nova-Pak C18 column (3.9xc3x97150 mm). Infrared spectra were recorded on a Nicolet Protxc3xa9gxc3xa9 460 FTIR as thin films or KBr pellets. 1HNMR spectra were recorded on either a Bruker AMX-400 or a Bruker ARX-500 NMR spectrometer and chemical shifts are expressed in parts per million (ppm or xcex4) with the solvent in use as internal standard. Coupling constants are given in hertz (Hz) and multiplets are designated as follows; singlet (s), doublet (d), triplet (t), quartet (q), muliplet (m), and broad (br). Low resolution mass spectra were determined on a Finnigan Matt TSQ-7000 triple stage quadrapole spectrometer (positive/negative ESI) operated in the negative ion mode.
Starting materials in the examples below may be synthesized by the methods disclosed in WO9807433, examples 1-106.